Preparation and characterization of a fully active biotinylated probe of cholecystokinin.
In this study we describe the synthesis and purification of biotinylated cholecystokinin-8 (Bio-CCK-8) and characterize its use as a probe for the pancreatic cholecystokinin receptor. CCK-8 (0.1 umoles) was reacted with either radiolabeled d-[8,9(-3)H]biotin succinimide ester (0.5 umoles) or N-hydroxysuccinimidyl-biotin in dimethylformamide and triethylamine, and purified by anion exchange chromatography. Concentrations of Bio-CCK-8 and CCK-8 needed for half-maximal inhibition of [125]I-CCK-8 binding to pancreatic membranes were the same (1.0 and 1.3 nM). Bio-CCK-8 retained full biological activity as determined by stimulation of pancreatic protein secretion from rats, and the biotin group bound to CCK-8 retained its high sensitivity for avidin.
Izzo
R S
RS
Department of Medicine, Nassau County Medical Center, East Meadow, New York 11554.
Pellecchia
C
C
Weinstein
M I
MI
Massimillo
A J
AJ
D000818:Animals; D001667:Binding, Competitive; D001710:Biotin; D002766:Cholecystokinin / Q000302:isolation & purification / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D010179:Pancreas / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D011949:Receptors, Cholecystokinin / Q000378:metabolism*; D013057:Spectrum Analysis
D011949:Receptors, Cholecystokinin; D001710:Biotin; D002766:Cholecystokinin